Astrocytic Activation Generates De Novo Neuronal Potentiation and Memory Enhancement.

Astrocytes respond to neuronal activity and were shown to be necessary for plasticity and memory. To test whether astrocytic activity is also sufficient to generate synaptic potentiation and enhance memory, we expressed the Gq-coupled receptor hM3Dq in CA1 astrocytes, allowing their activation by a designer drug. We discovered that astrocytic activation is not only necessary for synaptic plasticity, but also sufficient to induce NMDA-dependent de novo long-term potentiation in the hippocampus that persisted after astrocytic activation ceased. In vivo, astrocytic activation enhanced memory allocation; i.e., it increased neuronal activity in a task-specific way only when coupled with learning, but not in home-caged mice. Furthermore, astrocytic activation using either a chemogenetic or an optogenetic tool during acquisition resulted in memory recall enhancement on the following day. Conversely, directly increasing neuronal activity resulted in dramatic memory impairment. Our findings that astrocytes induce plasticity and enhance memory may have important clinical implications for cognitive augmentation treatments.

Hippocampal astrocytes encode reward location.

Astrocytic calcium dynamics has been implicated in the encoding of sensory information1-5, and modulation of calcium in astrocytes has been shown to affect behaviour6-10. However, longitudinal investigation of the real-time calcium activity of astrocytes in the hippocampus of awake mice is lacking. Here we used two-photon microscopy to chronically image CA1 astrocytes as mice ran in familiar or new virtual environments to obtain water rewards. We found that astrocytes exhibit persistent ramping activity towards the reward location in a familiar environment, but not in a new one. Shifting the reward location within a familiar environment also resulted in diminished ramping. After additional training, as the mice became familiar with the new context or new reward location, the ramping was re-established. Using linear decoders, we could predict the location of the mouse in a familiar environment from astrocyte activity alone. We could not do the same in a new environment, suggesting that the spatial modulation of astrocytic activity is experience dependent. Our results indicate that astrocytes can encode the expected reward location in spatial contexts, thereby extending their known computational abilities and their role in cognitive functions.

Dynamics of retrieval strategies for remote memories.

Prevailing theory suggests that long-term memories are encoded via a two-phase process requiring early involvement of the hippocampus followed by the neocortex. Contextual fear memories in rodents rely on the hippocampus immediately following training but are unaffected by hippocampal lesions or pharmacological inhibition weeks later. With fast optogenetic methods, we examine the real-time contribution of hippocampal CA1 excitatory neurons to remote memory and find that contextual fear memory recall, even weeks after training, can be reversibly abolished by temporally precise optogenetic inhibition of CA1. When this inhibition is extended to match the typical time course of pharmacological inhibition, remote hippocampus dependence converts to hippocampus independence, suggesting that long-term memory retrieval normally depends on the hippocampus but can adaptively shift to alternate structures. Further revealing the plasticity of mechanisms required for memory recall, we confirm the remote-timescale importance of the anterior cingulate cortex (ACC) and implicate CA1 in ACC recruitment for remote recall.

Molecular and cellular approaches for diversifying and extending optogenetics.

Optogenetic technologies employ light to control biological processes within targeted cells in vivo with high temporal precision. Here, we show that application of molecular trafficking principles can expand the optogenetic repertoire along several long-sought dimensions. Subcellular and transcellular trafficking strategies now permit (1) optical regulation at the far-red/infrared border and extension of optogenetic control across the entire visible spectrum, (2) increased potency of optical inhibition without increased light power requirement (nanoampere-scale chloride-mediated photocurrents that maintain the light sensitivity and reversible, step-like kinetic stability of earlier tools), and (3) generalizable strategies for targeting cells based not only on genetic identity, but also on morphology and tissue topology, to allow versatile targeting when promoters are not known or in genetically intractable organisms. Together, these results illustrate use of cell-biological principles to enable expansion of the versatile fast optogenetic technologies suitable for intact-systems biology and behavior.

Features of hippocampal astrocytic domains and their spatial relation to excitatory and inhibitory neurons.

The mounting evidence for the involvement of astrocytes in neuronal circuits function and behavior stands in stark contrast to the lack of detailed anatomical description of these cells and the neurons in their domains. To fill this void, we imaged >30,000 astrocytes in hippocampi made transparent by CLARITY, and determined the elaborate structure, distribution, and neuronal content of astrocytic domains. First, we characterized the spatial distribution of >19,000 astrocytes across CA1 lamina, and analyzed the morphology of thousands of reconstructed domains. We then determined the excitatory somatic content of CA1 astrocytes, and measured the distance between inhibitory neuronal somata to the nearest astrocyte soma. We find that on average, there are almost 14 pyramidal neurons per domain in the CA1, increasing toward the pyramidal layer midline, compared to only five excitatory neurons per domain in the amygdala. Finally, we discovered that somatostatin neurons are found in close proximity to astrocytes, compared to parvalbumin and VIP inhibitory neurons. This work provides a comprehensive large-scale quantitative foundation for studying neuron-astrocyte interactions.

Emerging technologies to study glial cells.

Development, physiological functions, and pathologies of the brain depend on tight interactions between neurons and different types of glial cells, such as astrocytes, microglia, oligodendrocytes, and oligodendrocyte precursor cells. Assessing the relative contribution of different glial cell types is required for the full understanding of brain function and dysfunction. Over the recent years, several technological breakthroughs were achieved, allowing "glio-scientists" to address new challenging biological questions. These technical developments make it possible to study the roles of specific cell types with medium or high-content workflows and perform fine analysis of their mutual interactions in a preserved environment. This review illustrates the potency of several cutting-edge experimental approaches (advanced cell cultures, induced pluripotent stem cell (iPSC)-derived human glial cells, viral vectors, in situ glia imaging, opto- and chemogenetic approaches, and high-content molecular analysis) to unravel the role of glial cells in specific brain functions or diseases. It also illustrates the translation of some techniques to the clinics, to monitor glial cells in patients, through specific brain imaging methods. The advantages, pitfalls, and future developments are discussed for each technique, and selected examples are provided to illustrate how specific "gliobiological" questions can now be tackled.

Sensory Deprivation Triggers Synaptic and Intrinsic Plasticity in the Hippocampus.

Hippocampus, a temporal lobe structure involved in learning and memory, receives information from all sensory modalities. Despite extensive research on the role of sensory experience in cortical map plasticity, little is known about whether and how sensory experience regulates functioning of the hippocampal circuits. Here, we show that 9 ± 2 days of whisker deprivation during early mouse development depresses activity of CA3 pyramidal neurons by several principal mechanisms: decrease in release probability, increase in the fraction of silent synapses, and reduction in intrinsic excitability. As a result of deprivation-induced presynaptic inhibition, CA3-CA1 synaptic facilitation was augmented at high frequencies, shifting filtering properties of synapses. The changes in the AMPA-mediated synaptic transmission were accompanied by an increase in NR2B-containing NMDA receptors and a reduction in the AMPA/NMDA ratio. The observed reconfiguration of the CA3-CA1 connections may represent a homeostatic adaptation to augmentation in synaptic activity during the initial deprivation phase. In adult mice, tactile disuse diminished intrinsic excitability without altering synaptic facilitation. We suggest that sensory experience regulates computations performed by the hippocampus by tuning its synaptic and intrinsic characteristics.

Neocortical excitation/inhibition balance in information processing and social dysfunction.

Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30-80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.

Global and local fMRI signals driven by neurons defined optogenetically by type and wiring.

Despite a rapidly-growing scientific and clinical brain imaging literature based on functional magnetic resonance imaging (fMRI) using blood oxygenation level-dependent (BOLD) signals, it remains controversial whether BOLD signals in a particular region can be caused by activation of local excitatory neurons. This difficult question is central to the interpretation and utility of BOLD, with major significance for fMRI studies in basic research and clinical applications. Using a novel integrated technology unifying optogenetic control of inputs with high-field fMRI signal readouts, we show here that specific stimulation of local CaMKIIalpha-expressing excitatory neurons, either in the neocortex or thalamus, elicits positive BOLD signals at the stimulus location with classical kinetics. We also show that optogenetic fMRI (of MRI) allows visualization of the causal effects of specific cell types defined not only by genetic identity and cell body location, but also by axonal projection target. Finally, we show that of MRI within the living and intact mammalian brain reveals BOLD signals in downstream targets distant from the stimulus, indicating that this approach can be used to map the global effects of controlling a local cell population. In this respect, unlike both conventional fMRI studies based on correlations and fMRI with electrical stimulation that will also directly drive afferent and nearby axons, this of MRI approach provides causal information about the global circuits recruited by defined local neuronal activity patterns. Together these findings provide an empirical foundation for the widely-used fMRI BOLD signal, and the features of of MRI define a potent tool that may be suitable for functional circuit analysis as well as global phenotyping of dysfunctional circuitry.

Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation.

Optogenetics with microbial opsin genes has enabled high-speed control of genetically specified cell populations in intact tissue. However, it remains a challenge to independently control subsets of cells within the genetically targeted population. Although spatially precise excitation of target molecules can be achieved using two-photon laser-scanning microscopy (TPLSM) hardware, the integration of two-photon excitation with optogenetics has thus far required specialized equipment or scanning and has not yet been widely adopted. Here we take a complementary approach, developing opsins with custom kinetic, expression and spectral properties uniquely suited to scan times typical of the raster approach that is ubiquitous in TPLSMlaboratories. We use a range of culture, slice and mammalian in vivo preparations to demonstrate the versatility of this toolbox, and we quantitatively map parameter space for fast excitation, inhibition and bistable control. Together these advances may help enable broad adoption of integrated optogenetic and TPLSMtechnologies across experimental fields and systems.

Reversible modulations of neuronal plasticity by VEGF.

Neurons, astrocytes, and blood vessels are organized in functional "neurovascular units" in which the vasculature can impact neuronal activity and, in turn, dynamically adjust to its change. Here we explored different mechanisms by which VEGF, a pleiotropic factor known to possess multiple activities vis-à-vis blood vessels and neurons, may affect adult neurogenesis and cognition. Conditional transgenic systems were used to reversibly overexpress VEGF or block endogenous VEGF in the hippocampus of adult mice. Importantly, this was done in settings that allowed the uncoupling of VEGF-promoted angiogenesis, neurogenesis, and memory. VEGF overexpression was found to augment all three processes, whereas VEGF blockade impaired memory without reducing hippocampal perfusion or neurogenesis. Pertinent to the general debate regarding the relative contribution of adult neurogenesis to memory, we found that memory gain by VEGF overexpression and memory impairment by VEGF blockade were already evident at early time points at which newly added neurons could not yet have become functional. Surprisingly, VEGF induction markedly increased in vivo long-term potentiation (LTP) responses in the dentate gyrus, and VEGF blockade completely abrogated LTP. Switching off ectopic VEGF production resulted in a return to a normal memory and LTP, indicating that ongoing VEGF is required to maintain increased plasticity. In summary, the study not only uncovered a surprising role for VEGF in neuronal plasticity, but also suggests that improved memory by VEGF is primarily a result of increasing plasticity of mature neurons rather than the contribution of newly added hippocampal neurons.

Analyzing engram reactivation and long-range connectivity.

Here, we present a protocol for marking engram cells to efficiently measure reactivation levels and their projection pathways. We describe steps for genetic manipulation utilizing transgenic mice and viral infections, labeling engram cells, and a modified version of CLARITY, a tissue-clearing technique. This protocol can be adapted to various research inquiries that involve assessing the overlap of cell populations and uncovering novel long-range connectivity pathways. For complete details on the use and execution of this protocol, please refer to Refaeli et al. (2023).1.

Astrocytes control recent and remote memory strength by affecting the recruitment of the CA1→ACC projection to engrams.

The maturation of engrams from recent to remote time points involves the recruitment of CA1 neurons projecting to the anterior cingulate cortex (CA1→ACC). Modifications of G-protein-coupled receptor pathways in CA1 astrocytes affect recent and remote recall in seemingly contradictory ways. To address this inconsistency, we manipulated these pathways in astrocytes during memory acquisition and tagged c-Fos-positive engram cells and CA1→ACC cells during recent and remote recall. The behavioral results were coupled with changes in the recruitment of CA1→ACC projection cells to the engram: Gq pathway activation in astrocytes caused enhancement of recent recall alone and was accompanied by earlier recruitment of CA1→ACC projecting cells to the engram. In contrast, Gi pathway activation in astrocytes resulted in the impairment of only remote recall, and CA1→ACC projecting cells were not recruited during remote memory. Finally, we provide a simple working model, hypothesizing that Gq and Gi pathway activation affect memory differently, by modulating the same mechanism: CA1→ACC projection.

VEGF is required for dendritogenesis of newly born olfactory bulb interneurons.

The angiogenic factor vascular endothelial growth factor A (VEGF) has been shown to have a role in neurogenesis, but how it affects adult neurogenesis is not fully understood. To delineate a role for VEGF in successive stages of olfactory bulb (OB) neurogenesis, we used a conditional transgenic system to suppress VEGF signaling at the adult mouse sub-ventricular zone (SVZ), rostral migratory stream (RMS) and OB, which constitute the respective sites of birth, the migration route, and sites where newly born interneurons mature and integrate within the existing OB circuitry. Following the development of fluorescently tagged adult-born neurons, we show that sequestration of VEGF that is constitutively expressed by distinct types of resident OB neurons greatly impaired dendrite development in incoming SVZ-born neurons. This was evidenced by reduced dendritic spine density of granule cells and significantly shorter and less branched dendrites in periglomerular neurons. Notably, the vasculature and perfusion of the SVZ, RMS and OB were not adversely affected when VEGF suppression was delayed until after birth, thus uncoupling the effect of VEGF on dendritogenesis from its known role in vascular maintenance. Furthermore, a requirement for VEGF was specific to newly born neurons, as already established OB neurons were not damaged by VEGF inhibition. This study thus uncovered a surprising perfusion-independent role of VEGF in the adult brain, namely, an essential role in the maturation of adult-born neurons.

Engram stability and maturation during systems consolidation.

Remote memories play an important role in how we perceive the world, and they are rooted throughout the brain in "engrams": ensembles of cells that are formed during acquisition. Upon their reactivation, a specific memory can be recalled.1,2,3,4,5,6,7,8,9,10,11,12 Many studies have focused on the ensembles in CA1 of the hippocampus and the anterior cingulate cortex (ACC). However, the evolution of these components during systems' consolidation has not yet been comprehensively addressed.13,14,15,16 By applying transgenic approaches for ensemble identification, CLARITY, retro-AAV, and pseudo-rabies virus for circuit mapping, and chemogenetics for functional interrogation, we addressed the dynamics of recent and remote CA1 ensembles. We expected both stability (as they represent the same memory) and maturation (over time). Indeed, we found that CA1 engrams remain stable between recent and remote recalls, and the inhibition of engrams for recent recall during remote recall functionally impairs memory. We also found that new cells in the remote recall engram in the CA1 are not added randomly during maturation but differ according to their connections. First, we show in two ways that the anterograde CA1 → ACC engram cell projection grows larger. Finally, in the retrograde projections, the ACC reduces input to CA1 engram cells, whereas input from the entorhinal cortex and paraventricular nucleus of the thalamus increases. Our results shine fresh light on systems' consolidation by providing a deeper understanding of engram stability and maturation in the transition from recent to remote memory.

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains.

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Mentoring: A three-generation perspective.

This NeuroView is intended for graduate students who are not sure how to choose or what to expect from a mentor as well as mentors who are uncertain what to give mentees. Two principal investigators and a current mentee will share their perspectives on this bidirectional relationship.

Immune modulation of learning, memory, neural plasticity and neurogenesis.

Over the past two decades it became evident that the immune system plays a central role in modulating learning, memory and neural plasticity. Under normal quiescent conditions, immune mechanisms are activated by environmental/psychological stimuli and positively regulate the remodeling of neural circuits, promoting memory consolidation, hippocampal long-term potentiation (LTP) and neurogenesis. These beneficial effects of the immune system are mediated by complex interactions among brain cells with immune functions (particularly microglia and astrocytes), peripheral immune cells (particularly T cells and macrophages), neurons, and neural precursor cells. These interactions involve the responsiveness of non-neuronal cells to classical neurotransmitters (e.g., glutamate and monoamines) and hormones (e.g., glucocorticoids), as well as the secretion and responsiveness of neurons and glia to low levels of inflammatory cytokines, such as interleukin (IL)-1, IL-6, and TNFα, as well as other mediators, such as prostaglandins and neurotrophins. In conditions under which the immune system is strongly activated by infection or injury, as well as by severe or chronic stressful conditions, glia and other brain immune cells change their morphology and functioning and secrete high levels of pro-inflammatory cytokines and prostaglandins. The production of these inflammatory mediators disrupts the delicate balance needed for the neurophysiological actions of immune processes and produces direct detrimental effects on memory, neural plasticity and neurogenesis. These effects are mediated by inflammation-induced neuronal hyper-excitability and adrenocortical stimulation, followed by reduced production of neurotrophins and other plasticity-related molecules, facilitating many forms of neuropathology associated with normal aging as well as neurodegenerative and neuropsychiatric diseases.

Astrocytes support hippocampal-dependent memory and long-term potentiation via interleukin-1 signaling.

Recent studies indicate that astrocytes play an integral role in neural and synaptic functioning. To examine the implications of these findings for neurobehavioral plasticity we investigated the involvement of astrocytes in memory and long-term potentiation (LTP), using a mouse model of impaired learning and synaptic plasticity caused by genetic deletion of the interleukin-1 receptor type I (IL-1RI). Neural precursor cells (NPCs), derived from either wild type (WT) or IL-1 receptor knockout (IL-1rKO) neonatal mice, were labeled with bromodeoxyuridine (BrdU) and transplanted into the hippocampus of either IL-1rKO or WT adult host mice. Transplanted NPCs survived and differentiated into astrocytes (expressing GFAP and S100ß), but not to neurons or oligodendrocytes. The NPCs-derived astrocytes from WT but not IL-1rKO mice displayed co-localization of GFAP with the IL-1RI. Four to twelve weeks post-transplantation, memory functioning was examined in the fear-conditioning and the water maze paradigms and LTP of perforant path-dentate gyrus synapses was assessed in anesthetized mice. As expected, IL-1rKO mice transplanted with IL-1rKO cells or sham operated displayed severe memory disturbances in both paradigms as well as a marked impairment in LTP. In contrast, IL-1rKO mice transplanted with WT NPCs displayed a complete rescue of the impaired memory functioning as well as partial restoration of LTP. These findings indicate that astrocytes play a critical role in memory functioning and LTP, and specifically implicate astrocytic IL-1 signaling in these processes. The results suggest novel conceptualization and therapeutic targets for neuropsychiatric disorders characterized by impaired astrocytic functioning concomitantly with disturbed memory and synaptic plasticity.

The memory orchestra: the role of astrocytes and oligodendrocytes in parallel to neurons.

For decades, the study of memory has been neuron-centric, yet neurons do not function in isolation. Today we know that neuronal activity is modulated by the environment within which it occurs, and is subject to modulation by different types of glial cells. In this review we summarize recent findings on the functional roles of astrocytes and oligodendrocytes, two major types of glia cells in the adult brain, in memory formation and its cellular underpinnings across multiple time points. We will discuss the different methods that are being used to investigate the astrocytic and oligodendroglial involvement in memory. We shall focus on chemogenetics and optogenetics, which support genetically specificity and high spatiotemporal resolution, attributes that are particularly well suited to the investigation of the contribution of unique cell types at the different stages of memory formation.